HIV replication fitness, which we will define as the growth rate of an HIV isolate relative to a reference HIV strain in vitro, has been proposed to be an important predictor of clinical outcome in HIV infection. The clinical significance of HIV replication fitness continues to be controversial, primarily because there is no consensus on the best way to measure relative replication fitness. Arguments persist as to whether fitness assays, using whole virus cultured from patient PBMCs, are preferable to those using recombinant viruses, and whether single cycle assays detect the same properties as multiple-cycle growth competition assays. We have developed a recombinant virus, multiple-cycle growth competition assay to measure HIV replication fitness in cell culture. In this assay, the proportion of infected cells is quantified using flow cytometry. We have modified this assay to measure relative replication fitness during a single cycle of virus replication, and will also modify the growth competition assay to measure replication fitness in intact ("whole") clinical isolates. These assays utilize similar viral vectors, and will quantify viral replication using the same methodology. We will then be in a unique position to evaluate how these fitness assays compare, and determine which of these approaches to measure replication fitness best correlates with clinical outcome. In order to achieve these goals we will accomplish the following specific aims: (1) Develop and evaluate a multiple-cycle recombinant-virus assay to measure HIV replication fitness in vitro. (2) Develop and evaluate a single-cycle recombinant-virus assay to measure HIV replication fitness in vitro. (3) Develop and evaluate a multiple-cycle whole-virus assay to measure HIV replication fitness in vitro. (4) Determine whether HIV-1 replication fitness, as measured using these three assays, correlates with viral load rebound during treatment failure or viral load set point after recent primary HIV infection. Relevance: The overall goal of our studies is to determine whether tests that measure how rapidly HIV grows can predict HIV viral load level. If these studies are successful, they could identify tests that could help predict clinical outcomes for HIV-infected patients.